Neutrophils are highly specialized cells in the host innate immune response that play a pivotal role in the clearance of bacterial infections and host inflammatory responses. Defects or overactivity in these cells can lead to life-threatening or debilitating diseases including sepsis, ischemia/reperfusion injury, rheumatoid arthritis, and chronic obstructive pulmonary disease (COPD). In addition, neutrophils are an excellent model for studying the mechanisms of cell polarization and directional cell migration for they are the fastest moving mammalian cells with overt cell polarity. Although a vast amount of work has been done, there are still many aspects of neutrophil biology that remain unclear. In addition, our gene expression analysis revealed a large number of proteins that are highly expressed in neutrophils, but with little knowledge about their roles in neutrophil biology. Unlike other phagocytes such as macrophages, primary differentiated neutrophils cannot be grown in culture for long and are not amenable to many of the in vitro manipulations. The majority of loss of function studies relies on targeted gene inactivation in mice or use neutrophil-like cells that difer from primary neutrophils in many regards and cannot be used for in vivo studies. To accelerate neutrophil research, we have developed a system that allows high expression of shRNAs in primary neutrophils in mice. The approach is amenable for a number of in vivo and in vitro assays for determining the importance of target proteins on various aspects of neutrophil biology. In this exploratory R21 proposal, we plan to screen targets that have no clear role in neutrophil biology or any biology. Our study will lead to identifying new proteins that play key roles in innate immunity and the inflammatory response and shed new lights into neutrophil biology, thus rapidly expanding the understanding of how neutrophils are regulated and undertake their cellular functions. Because many of the targets we propose to work on have homologs that are expressed in other tissues and cell type, our proposed studies may have potential impacts beyond neutrophils.